工程化OMEGA-TnpB系统的构建及体外靶向MDM2基因抑制骨肉瘤细胞恶性生物学行为的研究

Engineering OMEGA-TnpB system for in vitro targeted knockout of MDM2 gene to suppress malignant behaviors in osteosarcoma cells

  • 摘要:
    背景 传统治疗手段对晚期、复发及转移的骨肉瘤患者的治疗效果有限。基因治疗为骨肉瘤提供了新的治疗选择。然而,现有基因编辑工具(如CRISPR-Cas9)因尺寸大、免疫原性高等问题限制了其临床应用。新发现的OMEGA-TnpB系统具有紧凑的尺寸(约400 aa),在肿瘤基因编辑中展现出巨大潜力。目的 通过改造OMEGA-TnpB系统,开发高效、紧凑的基因编辑工具,并验证其在骨肉瘤基因治疗中的应用潜力。方法 通过理性设计获取 TnpB 突变体。利用单链退火(SSA)报告系统筛选突变体,并在HEK293T和骨肉瘤细胞系(143B)中验证其编辑效率。最后使用优化的TnpB突变体敲除骨肉瘤基因MDM2,通过RT-qPCR、流式细胞术、CCK-8、划痕实验和Transwell实验评估其对细胞增殖、凋亡、迁移及侵袭的影响。结果 筛选出多个高活性TnpB突变体,其中ISYmu1-L225K在HEK293T(P<0.01)和143B细胞(P<0.01)中的编辑效率显著高于enOsCas12f1,在HEK293T细胞部分位点与SpCas9相比无显著差异。敲除MDM2基因后,骨肉瘤细胞的增殖能力下降(P<0.01),凋亡率增加(P<0.01),迁移(P<0.01)和侵袭(P<0.05)能力被抑制。结论 本研究成功获取了在哺乳动物细胞内基因编辑效率提高的TnpB突变体ISYmu1-L225K,验证了ISYmu1-L225K能靶向敲除MDM2基因并抑制骨肉瘤细胞的恶性行为,为骨肉瘤的精准治疗提供了新思路。

     

    Abstract:
    Background Conventional treatments have limited effectiveness in patients with advanced, recurrent, or metastatic osteosarcoma. Gene therapy offers a promising therapeutic option for osteosarcoma. However, the clinical application of current gene-editing tools (e.g. CRISPR-Cas9) is limited by their large size and high immunogenicity. The newly discovered OMEGA-TnpB system, with its compact size (~ 400 aa), shows great potential for tumor gene editing.Objective To develop efficient and compact gene-editing tools by engineering the OMEGA-TnpB system, and validate their potential for osteosarcoma gene therapy.Methods Through rational design, TnpB mutants were obtained. A single-strand annealing (SSA) reporter system was employed to screen mutants, and their editing efficiency was validated in HEK293T and osteosarcoma cell lines (143B). The optimized TnpB mutant (ISYmu1-L225K) was used to knock out the osteosarcoma gene MDM2. The effects on cell proliferation, apoptosis, migration, and invasion were assessed by RT-qPCR, flow cytometry, CCK-8, wound-healing assay, and Transwell assay.Results Several highactivity TnpB mutants were identified, among which ISYmu1-L225K exhibited significantly higher editing efficiency than enOsCas12f1 in both HEK293T (P<0.01) and 143B cells (P<0.01). In HEK293T cells, its efficiency at certain loci showed no significant difference compared to SpCas9. Knockout of MDM2 reduced osteosarcoma cell proliferation (P<0.01), increased apoptosis rates (P<0.01), and inhibited both migration (P<0.01) and invasion (P<0.05). Conclusion This study successfully develops the TnpB mutant ISYmu1-L225K, which exhibits enhanced gene editing efficiency in mammalian cells. ISYmu1-L225K suppresses the malignant behavior of osteosarcoma cells by targeting MDM2, providing a novel strategy for precision therapy in osteosarcoma.

     

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